The overarching goal of this project is to evaluate the role of reciprocal regulation between EphA2 and Akt in glioma invasion and to understand the underlying molecular mechanisms. Glioblastoma multiforme (GBM) is the most common primary brain tumor. It is an incurable malignancy due in large part to its diffuse invasion. Recent large scale genome studies show that PI3K/Akt signaling cascade is activated in 88% of GBM. While Akt is well known to control cell proliferation and survival, its role in cell migration and invasion is not well understood. We discovered that Akt promotes glioma cell invasion by targeting EphA2. As a member of Eph subfamily of receptor tyrosine kinases, EphA2 has been extensively studied in cancer. Paradoxically, both pro- and anti-oncogenic functions have been attributed to EphA2. We reported recently that EphA2 has diametrically opposite roles in regulating glioma cell migration and invasion. In the presence of ligands called ephrin-As, EphA2 inhibited cell migration and invasion. In contrast, in the absence of ligands, EphA2 promoted chemotactic migration and invasion instead. Interestingly, the ligand-independent stimulation of cell motility was correlated with phosphorylation of EphA2 on serine 897 by Akt. S897A mutation abolished this ligand-independent effect. In human glioma specimens, S897 phosphorylation is correlated with tumor grades and Akt activation, suggesting pathological relevance. The data in aggregate suggest that the Akt-EphA2 signaling axis contributes to invasion of glioma. However, direct proof in vivo has not been demonstrated yet, nor is it known how Akt/EphA2 crosstalk promotes cell migration and invasion at the molecular level. The goal of this proposal is to fill both gaps. In Specific Aim 1, we will determine whether the Akt-EphA2 signaling axis promotes human GBM invasion in vivo. Aim 2 will test whether deletion of ligands for EphA2 will lead to accelerated glioma invasion. In Aim 3, we will characterize the molecular and structural bases underlying stimulation of cell migration and invasion by the Akt-EphA2 signaling axis. PUBLIC HEALTH RELEVANCE: Despite maximum treatments, the prognosis of GBM is dismal with median survival of only 14 months. The inevitable lethality is in large part caused by the diffuse infiltrative invasion throughout the brain at the time of diagnosis. Understanding molecular mechanisms of GBM invasion is central in developing new therapeutic strategies. We have characterized a new molecular pathway that constitutes an "on/off" switch for glioma cell migration in vitro. The proposed studies will extend this important discovery in a test tube to preclinical models in vivo and investigate the molecular and structural bases of the switch using several highly innovative approaches and model systems. As such, the current application has highly significant public health relevance, because it will not only inform us whether the switch works in vivo, but also reveal components of glioma invasion machinery. The information can lead to the development of new agents for treating this deadly disease.